One family of diagnostic assays that has become well known and accepted in the field of clinical chemistry is based upon nucleic acid hybridization. It is well known that nucleic acid molecules (DNA and RNA) can and do hybridize to complementary strands of nucleic acids, thereby forming double stranded molecules.
The principles of nucleic acid hybridization is built upon two deceptively simple rules. All nucleic acid molecules consist of four nucleotide bases DNA contains bases "A", "T", "C" and "G", whereas RNA contains "A", "U", "C" and "G". The two simple rules referred to supra are (i) the bases "A" and "T" and "U" complement and hybridize to each other, and "C" and "G" to each other. Nucleic acid molecules of varied and great lengths can be formed via the binding of bases to each other.
The ability to identify particular organisms, viruses, mutations in DNA/RNA sequences, etc., relies on the presence of sequences unique to the material to be identified, and the hybridization principles referred to supra. For example, if microorganism "X" contains unique DNA sequence: EQU 5'-AAACCGGCC-3'
it is theoretically possible to identify the microorganism by making the unique sequence accessible and contacting it by its complement: EQU 3'-TTTGGCCGG-5
If the complement contains a label, i.e., some "marker" it will be possible to note that hybridization has occurred. It is this principle that is the core of all nucleic acid determination assays.
The strand displacement assay is generally described in a series of U.S. Patents, i.e. U.S. Pat. Nos. 4,629,689 (Diamond et al.), 4,725,536 (Fritsch et al.), 4,725,537 (Fritsch et al.), 4,735,897 (Vary et al.), 4,752,566 (Collins et al.), 4,766,062 (Diamond et al.), 4,766,064 (Williams et al.), 4,767,699 (Vary et al.), 4,795,701 (Vary), 4,818,680 (Collins et al.). Essentially, it involves the contacting of a test sample believed to contain a nucleic acid sequence of interests with a hybrid complex of two nucleic acid sequences of differing lengths. The longer sequence is referred to as the binding probe, and is complementary to the nucleic acid sequence of interest. A shorter sequence, referred to as the signal probe, is hybridized to only a portion of the binding probe. That portion of the binding probe which is not hybridized to signal probe is referred to as the initial binding region. For example, using the sequence example given supra: EQU 5'-AAACCGGCC-3'
a hybrid complex such as the following may be prepared: EQU TTGGCCGG EQU GGCC
wherein "TTGGCCGG" is the binding probe, "TTGG" is the initial binding region, and "GGCC" is the signal probe. In practice, the binding probe binds to the target sequence via the initial binding region, or "IBR". The binding probe continues to hybridize to the target molecule, because it is longer than the signal probe, and complexes which include longer molecules are more stable. Nucleic acids do not form stable triplex complexes, however, and thus the signal probe is "displaced". When the signal probe is labelled, this displacement can be measured.
The strand displacement assay is useful, but is not without its problems, not the least of which is sensitivity. Pathological conditions are known, e.g., where a difference between normality and abnormality rests in a single nucleotide base difference. Sickle cell anemia is simply the most well known example of a pathological condition identified by this type of point mutation. There are other analytes where determination of a single nucleotide base is important, or where differences between various microorganisms is slight. Identification and determination in such contexts requires levels of sensitivity and specificity not previously obtainable with strand displacement assays.
One organism which has been studied quite extensively in connection with nucleic acid assays is Neisseria gonorrhoeae, the causative agent of a sexually transmitted disease, with an estimated two million reported cases per year. Examples of the patent literature regarding N. gonorrhoeae specific nucleic acid probes include U.S. Pat. Nos. 4,900,659 to Lo et al, and 5,047,523 and 5,099,011 both to Woods et al. These patents deal with DNA:DNA hybridization, and disclose probes which, while useful, are simply not very specific.
Additionally, published patent applications WO90/14442, EPA 337,896, EPA 272009 and EPA 408077 disclose hybridization between RNA and complementary probes. These patents represent developments on the patent of Kohne, U.S. Pat. No. 4,851,330, which identified organisms by detecting unique RNA sequences, in that the rRNA sequence of the organism is the target.
None of these references teach the specific nucleic acid probes for N. gonorrhoeae, disclosed herein, which are useful in various diagnostic assays including the strand displacement assay. Not only are the sequences disclosed herein unique, they function as specific N. gonorrhoeae probes. With respect to this statement, it must be pointed out that uniqueness of a specific sequence does not necessarily mean that the sequence will function as a probe. Various considerations, including the total length of the probe, the ability to identify large numbers of strains of the organism, micro heterogeneity, etcetera must be considered.
In view of the difficulty of identifying probes which are useful as N. gonorrhoeae probes, it is surprising that probes which vary in only a single base pair from comparable sequences of different species of Neisseria can be used to identify N. gonorrhoeae. Such probes are another feature of the invention as described herein.